In vitro selection of DNA aptamers that bind L-tyrosinamide

We have applied SELEX (Systematic Evolution of Ligands by EXponential enrichment), a combinatorial method that employs biopolymers for drug discovery, to identify single stranded DNA sequences able to bind L-Tyrosinamide, a simple mimic of Tyrosine, an amino acid essential to the catalytic activity of several enzymes of pharmaceutical interest. After 15 SELEX cycles using L-Tyrosinamide immobilized on an affinity chromatography column, the percentage of aptamers specifically eluted from the affinity column with free L-Tyrosinamide was 55% of the total. Aptamers were subcloned and sequenced, allowing the identification of a highly conserved consensus sequence, and showed a K(d) value for L-Tyrosinamide of 45 microM. The identified aptamer sequence will constitute the basis for further in vitro evolution protocols and structure-based drug design.     

In vitro selection of single-stranded DNA aptamers that bind human pro-urokinase

Single-chain pro-urokinase is an inactive proenzyme form of human urokinase (urinary plasminogen activator) with a Mr of 50,000 which is converted to the active two-chain form by catalytic amounts of plasmin. It is used for thrombolytic therapy of acute myocardial infarction and acute ischemic stroke. We have isolated single-stranded DNA molecules with significantly increased binding affinity for human pro-urokinase by SELEX (systematic evolution of ligands by exponential enrichment) procedure from a pool of 10(15) molecules containing 24 randomized positions which are flanked by defined regions. ssDNA from this library was hybridized with helper "fixture", thus allowing the central random chain to fold into complex three-dimentional shapes. Sequencing data from pro-urokinase aptamers obtained after 12 selection cycles displayed a highly conserved 12-14 base region.     

In vitro selection of DNA aptamers binding ethanolamine

We have identified aptamers (synthetic oligonucleotides) binding to the very small molecule ethanolamine with high affinity down to the low nanomolar range. These aptamers were selected for their ability to bind to ethanolamine immobilised on magnetic beads, from an 96mer library of initially about 1 x 10(16) randomised ssDNA molecules. The dissociation constants of these aptamers range between K(D)=6 and K(D)=19 nmol L(-1). The aim of the development of ethanolamine aptamers is their use for the detection of this substance in clinical and environmental analysis. Ethanolamine is associated with several diseases. Moreover, ethanolamine and its derivatives di- and tri-ethanolamine are used in chemical and cosmetic industries. The use of biosensors with ethanolamine aptamer as new molecular recognition element could be an innovative method for an easy and fast detection of ethanolamine.     

In vitro selection of hematoporphyrin binding DNA aptamers

DNA aptamers that bind to hematoporphyrin IX (HPIX) were isolated using an in vitro selection technique. Most aptamers obtained after the 7th and 10th rounds contained guanine-rich sequences. Binding assay using fluorescence polarization technique and structural analysis by CD spectra revealed that the parallel guanine-quartet structure of the aptamer participates in the recognition of HPIX.     

In vitro selection and characterization of cellulose-binding DNA aptamers

Many nucleic acid enzymes and aptamers have modular architectures that allow them to retain their functions when combined with other nucleotide sequences. This modular function facilitates the engineering of RNAs and DNAs that have more complex functions. We sought to create new DNA aptamers that bind cellulose to provide a module for immobilizing DNAs. Cellulose has been used in a variety of applications ranging from coatings and films to pharmaceutical preparations, and therefore DNA aptamers that bind cellulose might enable new applications. We used in vitro selection to isolate aptamers from a pool of random-sequence DNAs and subjected two distinct clones to additional rounds of mutagenesis and selection. One aptamer (CELAPT 14) was chosen for sequence minimization and more detailed biochemical analysis. CELAPT 14 aptamer variants exhibit robust binding both to cellulose powder and paper. Also, an allosteric aptamer construct was engineered that exhibits ATP-mediated cellulose binding during paper chromatography.     

In vitro selection of DNA aptamers binding pesticide fluoroacetamide

Fluoroacetamide (Mw = 77.06) is a lethal rodenticide to humans and animals which is still frequently abused in food storage somewhere in China. The production of antibodies for fluoroacetamide is difficult due to its high toxicity to animals, which limits the application of immunoassay method in poison detection. In this work, aptamers targeting N-fluoroacetyl glycine as an analog of fluoroacetamide were selected by a specific systematic evolution of ligands by exponential enrichment (SELEX) strategy. The binding ability of the selected aptamers to fluoroacetamide was identified using surface plasmon resonance (SPR)-based assay. The estimated K_D values in the low micromolar range showed a good affinity of these aptamers to the target. Our work verified that the SELEX strategy has the potential for developing aptamers targeted to small molecular toxicants and aptamers can be employed as new recognition elements instead of antibodies for poison detection.     

In vitro selection of high-affinity DNA aptamers for streptavidin

In this study, we developed a systematic evolution of ligands by exponential enrichment （SELEX） method using a combination of magnetic beads immobilization and flow cytometric measurement. As an example, the selection of streptavidin-specific aptamers was performed. In this protocol, the conventional SELEX procedure was optimized, fiirst using magnetic beads for target immobilization to facilitate highly efficient separation of the binding single-stranded DNA （ssDNA） aptamers from the unbound ssDNAs, and second using flow cytometry and fluorescein labeling to monitor the enrichment. The sensitivity of flow cytometry was adequate for ssDNA quantification during the SELEX procedures. The streptavidin-specific aptamers obtained in this work can be used as tools for characterization of the occupancy of streptavidin-modified surfaces with biotinylated target molecules. The method described in the study is also generally applicable to target molecules other than streptavidin.     

In vitro selection and characterization of DNA aptamers recognizing chloramphenicol

Chloramphenicol (Cam), although an effective antibiotic, has lost favour due to some fatal side effects. Thus there is an urgent need for rapid and sensitive methods to detect residues in food, feed and environment. We engineered DNA aptamers that recognize Cam as their target, by conducting in vitro selections. Aptamers are nucleic acid recognition elements that are highly specific and sensitive towards their targets and can be synthetically produced in an animal-friendly manner, making them ethical innovative alternatives to antibodies. None of the isolated aptamers in this study shared sequence homology or structural similarities with each other, indicating that specific Cam recognition could be achieved by various DNA sequences under the selection conditions used. Analyzing the binding affinities of the sequences, demonstrated that dissociation constants (K_d) in the extremely low micromolar range, which were lower than those previously reported for Cam-specific RNA aptamers, were achieved. The two best aptamers had G rich (>35%) nucleotide regions, an attribute distinguishing them from the rest and apparently responsible for their high selectivity and affinity (K_d ∼ 0.8 and 1 μM respectively). These aptamers open up possibilities to allow easy detection of Cam via aptamer-based biosensors.     

In vitro selection of signaling aptamers

Reagentless biosensors that can directly transduce molecular recognition to optical signals should potentiate the development of sensor arrays for a wide variety of analytes. Nucleic acid aptamers that bind ligands tightly and specifically can be readily selected, but may prove difficult to adapt to biosensor applications. We have therefore attempted to develop selection methods that couple the broad molecular recognition properties of aptamers with signal transduction. Anti-adenosine aptamers were selected from a pool that was skewed to contain very few fluoresceinated uridines. The primary family of aptamers showed a doubling of relative fluorescence intensity at saturating concentrations of a cognate analyte, ATP, and could sense ATP concentrations as low as 25 microM. A single uridine was present in the best signaling aptamer. Surprisingly, other dyes could substitute for fluorescein and still specifically signal the presence of ATP, indicating that the single uridine functioned as a general "switch" for transducing molecular recognition to optical signals.     

In vitro selection of DNA aptamers to anthrax spores with electrochemiluminescence detection.

Systematic evolution of ligands by exponential enrichment (SELEX) was used to select and PCR amplify DNA sequences (aptamers) capable of binding to and detecting nonpathogenic Sterne strain Bacillus anthracis spores. A simplified affinity separation approach was employed, in which autoclaved anthrax spores were used as the separation matrix. An aptamer-magnetic bead-electrochemiluminescence (AM-ECL) sandwich assay scheme was devised for detecting anthrax spores. Using a low SELEX DNA to spore ratio (154 ng DNA/10(6) spores), at least three distinct populations of single-stranded DNA aptamers, having varied affinities for anthrax spores, were noted by the AM-ECL assay. Results reflect detection of spore components with a dynamic range equivalent to < 10- > 6 x 10(6) anthrax spores. In the low DNA to spore ratio experiments, aptamers could be liberated from spore pellets by heating at 96 degrees C for 5 min after each round of SELEX. When a much higher DNA to spore ratio (10,256 ng DNA/10(6) spores) was used for SELEX development, a higher affinity set of aptamers was selected that could not be heat-eluted even at 99 degrees C for 5 min following round four of SELEX. However, high affinity spore surface bound aptamers were detectable via their 5'-biotinylated tails using labeled avidin and could be eluted in deionized water. Aptamers have potential for use as inexpensive, in vitro-generated receptors for biosensors in biological warfare detection and other areas.     

DNA aptamers that bind to chitin

We have succeeded in the acquisition of DNA aptamers that recognize chitin using in vitro selection. The obtained DNA aptamers have the stem-loop or bulge loop structures with guanine rich loop clusters and the clockwise B-form stems.     

In vitro selection of RNA aptamers that bind to cell adhesion receptors of Trypanosoma cruzi and inhibit cell invasion

Trypanosoma cruzi causing Chagas' disease needs to invade host cells to complete its life cycle. Macromolecules on host cell surfaces such as laminin, thrombospondin, heparan sulfate, and fibronectin are believed to be important in mediating parasite-host cell adhesions and in the invasion process of the host cell by the parasite. The SELEX technique (systematic evolution of ligands by exponential enrichment) was used to evolve nuclease-resistant RNA ligands (aptamer = to fit) that bind with affinities of 40-400 nm to parasite receptors for the host cell matrix molecules laminin, fibronectin, thrombospondin, and heparan sulfate. After eight consecutive rounds of in vitro selection four classes of RNA aptamers based on structural similarities were isolated and sequenced. All members of each class shared a common sequence motif and competed with the respective host cell matrix molecule that was used for displacement during the selection procedure. RNA pools following seven and eight selection rounds as well as individual aptamers sharing consensus motifs were active in inhibiting invasion of LLC-MK(2) monkey kidney cells by T. cruzi in vitro.     

In vitro selection of specific RNA aptamers for the NFAT DNA binding domain

Nuclear factor of activated T cells (NFAT) plays a central role in the immune response, and the immuno-suppressive drugs, cyclosporin A and FK-506, have been developed to inhibit it. However, due to the toxic effects of these drugs, which derive from their ability to inhibit calcineurin in non-immune tissues, the identification of small compounds that target NFAT directly could be an approach to developing less toxic immunosuppressive therapy. Using an in vitro selection technology termed SELEX on a combinatorial RNA library with 40 nucleotide-long random sequences, we have isolated two RNA aptamers to the NFAT DNA binding domain (DBD). Gel retardation assays and surface plasmon resonance measurements showed that the aptamers have a specific and high affinity (apparent KD~10 to 100 nM) for the NFAT DBD. Enzymatic probing analysis showed that the two RNA aptamers have similar structures and share a sequence that forms an apical loop. Moreover, RNase footprinting analysis showed that the shared sequence (GATATGAAGGA/ TGTG/AGAGAG) is critical for binding to both NFATp DBD and NFATc DBD. These results suggest that short RNAs identified in this study is a specific aptamer to NFAT DBD, and hence could be applied not only for the delineation of NFAT functions but for the development of potent immune modulating lead compounds.     

In vitro selection of DNA aptamers against the HIV-1 TAR RNA hairpin

In vitro selection was performed to identify DNA aptamers against the TAR RNA stem-loop structure of HIV-1. A counterselection step allowed the elimination of kissing complex-forming aptamers previously selected (Boiziau et al. J. Biol. Chem. 1999; 274:12730). This led to the emergence of oligonucleotides, most of which contained two consensus sequences, one targeted to the stem 3'-strand (5'-CCCTAGTTA) and the other complementary to the TAR apical loop (5'-CTCCC). The best aptamer could be shortened to a 19-mer oligonucleotide, characterized by a dissociation constant of 50 nM. A 16-mer oligonucleotide complementary to the TAR stem 3'-strand could also be derived from the identified aptamers, with an equal affinity (Kd = 50 nM). Experiments performed to elucidate the interaction between TAR and the aptamers (UV melting measures, enzymatic and chemical footprints) demonstrated that the TAR stem 5'-strand was not simply displaced as a result of the complex formation but unexpectedly remained associated on contact with the antisense oligonucleotide. We suggest that a multistranded structure could be formed.     

Selection of DNA aptamers that bind to four organophosphorus pesticides

Single-stranded DNA (ssDNA) aptamers against four organophosphorus pesticides (phorate, profenofos, isocarbophos and omethoate) were simultaneously isolated from an immobilized random ssDNA library by systematic evolution of ligands by exponential enrichment (SELEX) technique. After 12 rounds of in vitro selection, five ssDNA aptamer candidates were selected and their binding affinities were identified by a novel method using a molecular beacon. Two of the five ssDNA sequences, SS2-55 and SS4-54, demonstrated higher affinities and specificities to the four organophosphorus pesticides. They were defined as broad-spectrum aptamers binding to four different targets and their simulated secondary structures showed highly distinct features with typical stem and loop structures. The dissociation constant of SS2-55 and SS4-54 binding to the four organophosphorus pesticides ranged from 0.8 to 2.5?μM. These aptamers offered application potential in the analysis and/or neutralization of the residues of the four organophosphorus pesticides.     

In vitro selection of RNA aptamers that bind to colicin E3 and structurally resemble the decoding site of 16S ribosomal RNA

Colicin E3 is a ribonuclease that specifically cleaves at the site after A1493 of 16S rRNA in Escherichia coli ribosomes, thus inactivating translation. To analyze the interaction between colicin E3 and 16S rRNA, we used in vitro selection to isolate RNA ligands (aptamers) that bind to the C-terminal ribonuclease domain of colicin E3, from a degenerate RNA pool. Although the aptamers were not digested by colicin E3, they specifically bound to the protein (K(d) = 2-14 nM) and prevented the 16S rRNA cleavage by the C-terminal ribonuclease domain. Among these aptamers, aptamer F2-1 has a sequence similar to that of the region around the cleavage site from residue 1484 to 1506, including the decoding site, of E. coli 16S rRNA. The secondary structure of aptamer F2-1 was determined by the base pair covariation among the variants obtained by a second in vitro selection, using a doped RNA pool based on the aptamer F2-1 sequence. The sequence and structural similarities between the aptamers and 16S rRNA provide insights into the recognition of colicin E3 by this specific 16S rRNA region.     

In vitro selection of oligonucleotides that bind double-stranded DNA in the presence of triplex-stabilizing agents

A SELEX approach has been developed in order to select oligonucleotides that bind double-stranded DNA in the presence of a triplex-stabilizing agent, and was applied to a target sequence containing an oligopurine–oligopyrimidine stretch. After only seven rounds of selection, the process led to the identification of oligonucleotides that were able to form triple helices within the antiparallel motif. Inspection of the selected sequences revealed that, contrary to GC base pair which were always recognized by guanines, recognition of AT base pair could be achieved by either adenine or thymine, depending on the sequence context. While thymines are strongly preferred for several positions, some others can accommodate the presence of adenines. These results contribute to set the rules for designing oligonucleotides that form stable triple helices in the presence of triplex-stabilizing agents at physiological pH. They set the basis for further experiments regarding extension of potential target sequences for triple-helix formation or recognition of ligand–DNA complexes.